Intraamniotic Infection of Midtrimester Amniocentesis: A ...

Infectious Diseases in Obstetrics and Gynecology 2:136-139 (1994)(C) 1994 Wiley-Liss, Inc.

Occult Intraamniotic Infection at the Time ofMidtrimester Genetic Amniocentesis:

A Reassessment

Peter H. Cherouny, Glenn A. Pankuch, and John J. BottiDepartment of Obstetrics and Gynecology, University of Vermont, Burlington, VT (P.H.C.), andDepartments of Obstetrics and Gynecology (J.J.B.) and Pathology (G.A.P.), Pennsylvania State

University, Hershey, PA

ABSTRACT

Objective: The objective of this study was to reevaluate the incidence of occult early midtrimesterintraamniotic infection in asymptomatic patients at the time of genetic amniocentesis.

Methods: A total of 177 amniotic fluid (AF) specimens from patients referred for genetic amnio-centesis between 15 and 20 postmenstrual weeks were evaluated for the presence of bacteria bydetailed light microscopy, after Gram and Wright stain, and by cultures for aerobic and anaerobicbaceria, Mycoplasma sp., and Ureaplasma urealyticum. Seventy-seven AF specimens were also testedfor the presence of bioactive leukoattractants by a leukotaxis bioassay.

Results: All fluids were negative for bacteria and bioactive leukoattractants [95% confidenceinterval (CI), 0-1.9%; 99% CI, 0-2.9%]. This is significantly less than a recently reported incidenceof 5.09% (P 0.002). Incidentally, artifacts with light microscopic morphology consistent withspermatozoa were found during the detailed light microscopic evaluation ofAF Gram stains from 2(1.1%) AF samples in otherwise uneventful pregnancies, a previously unreported finding. Scanningelectron microscopy was used to confirm the light microscopic findings.

Conclusions: Occult intraamniotic infection in the second trimester is not as high as recentlyreported. AF culture in all cases of second-trimester amniocentesis is not necessary. The identifica-tion of spermatozoa on Gram stain of second-trimester AF specimens needs further confirmation.(C) 1994 Wiley-Liss, Inc.

KEY WORDS

Amniotic fluid, mycoplasmas, leukoattractants

has been well established that bacteria and bioac-tive leukoattractants may be identified in amniotic

fluid (AF) when chorioamniotic membranes are

intact. ,2 Positive AF cultures in patients in idio-pathic preterm labor with intact membranes have a

reported incidence of 4-26%. The most frequentlyisolated organisms in this setting include Fusobacte-rium nucleatum, Mycoplasma sp., and Ureaplasfnaurealyticum. Positive AF cultures are almost uni-versally associated with failed tocolysis and deliverysoon after AF collection. The presence of AF leu-koattractants determined by the leukotaxis bioassay

is also a sensitive marker for AF infection, pretermdelivery, and histologic chorioamnionitis in the pre-term-labor population. 3 The leukotaxis assay is sen-sitive to a wide range and concentration of leukotac-tic factors and detects biologically active forms ofleukoattractants. This is an advantage over immu-nologic assays of specific leukoattractants whichidentify immunoactive forms without addressingbioactivity.

The incidence of occult AF infection at the timeof genetic amniocentesis in the early second trimes-ter is not as well established. It has been recently

Address correspondence/reprint requests to Dr. Peter H. Cherouny, Medical Center Hospital of Vermont, Shepardson 335,Burlington, VT 05401.

Received February 15, 1994Clinical Study Accepted July I, 1994

OCCULT MIDTRIMESTER INTRAAMNIOTIC INFECTION CHEROUNY ET AL.

reported as >5% (8/157), more than 10 timeshigher than the loss risk associated with amniocen-tesis. 4 The organisms identified in this previousreport are not commonly isolated from AF col-lected from patients with preterm labor. Specifi-cally, no anaerobic bacteria were identified andcultures for genital mycoplasms were not per-formed. The present study was, therefore, under-taken to reevaluate the incidence of occult infectionof the amniotic cavity at the time of midtrimestergenetic amniocentesis.

SUBJECTS AND METHODSAF was collected from 177 patients with viablesingleton pregnancies, without identifiable abnor-malities, referred for midtrimester amniocentesisbetween 15 and 20 weeks of gestation to UniversityHospital of the Pennsylvania State UniversitySchool of Medicine. Informed consent was obtainedin order to use an aliquot of AF for the study. Theindications for amniocentesis included advancedmaternal age (89%), low maternal serum alphafeto-protein (6%), parental anxiety (1%), and history ofchromosomal abnormality in a prior pregnancy(4%). No patient related a history consistent withruptured amniotic membranes and all had normalAF on ultrasound. Amniocentesis was perfo’medby one of the authors (P.H.C. or j.j.B.), underultrasound guidance and with strict asepsis, using a

20-gauge, 88.9-mm spinal needle.AF was evaluated for bacteria by Gram stain,

Wright stain, aerobic and anaerobic cultures, andculture for Mycoplasma sp. and U. urealyticum, as

previously described. 2 In brief, AF was sent to theresearch laboratory for processing within h ofcollection. When delays of > h but < 12 h were

anticipated, a portion of the sample was inoculatedinto a Port-A-Cul vial (BBL Microbiology Sys-tems, Cockeysville, MD) for later culture and stain-ing. The remaining fluid was refrigerated at 4Cuntil processed for Mycoplasma culture and, if fluidwas available, for leukotaxis assay.

After Gram staining, AF was cultured for aero-

bic and anaerobic bacteria by spreading 0.1-, 0.01-,and 0.001-ml aliquots of uncentrifuged AF induplicate on the following media: trypticase soyagar with 5% sheep blood (BBL Microbiology Sys-tems); chocolate agar (BBL Microbiology Systems);prereduced chopped-meat glucose broth (ScottLaboratories, Fiskeville, RI); enriched brucella

blood and laked blood agars (incubated anaerobi-cally).

After culturing, AF was centrifuged at 1,000gfor 10 min. After Wright staining, the pellet was

cultured for M. hominis and U. urealyticum in thefollowing manner. Ten microliters ofthe pellet wasinoculated into urease color-test broth U9C. Three10-fold serial dilutions were prepared in the U9Cbroth (incubated aerobically at 37C for 21 days).Ten microliters from the pellet and 10 p,1 fromeach serial dilution were spread onto A8 differen-tial agar medium (incubated anaerobically).An aliquot of AF, available from 77 samples

after culturing, was frozen at -70C until evalua-tion for bioactive leukoattractants by the leukotaxisassay, as previously described. 3 No change in leu-koattractant activity has been noted for samplesstored in this manner for up to 2 years. Statisticalanalysis was performed using Fisher’s exact testwith confidence intervals (CIs) determined on per-centages by Poisson distribution. A value of P <0.05 was considered statistically significant.

RESULTSAll bacteriologic studies were negative on all fluidspecimens (0-1.9% and 0-2.9%, 95% and 99%CIs, respectively). In addition, io bioactive leuko-attrac’tants were identified in the AF aliquots avail-able for testing with the leukotaxis bioassay. Therewere no immediate or delayed complications associ-ated with amniocentesis. Specifically, no pregnancylosses occurred within 4 weeks of amniocentesis.Gram stain from 2 AF samples was observed to

have artifacts with light microscopic morphologiccharacteristics consistent with spermatozoa, a find-ing not previously reported (Fig. 1). Scanning elec-tron microscopy was used to confirm the presenceof spermatozoa on the Gram-stained specimens(Figs. 2-4). These AF specimens did not containbacteria or bioactive leukoattractants. Both fetuseswere male.

DISCUSSIONThis study did not find bacterial colonization in theearly second trimester in patients presenting forgenetic amniocentesis. Furthermore, this studyfailed to show genital mycoplasmas or bioactiveleukoattractants in any AF specimen tested in addi-tion to negative aerobic and anaerobic AF culturesin asymptomatic patients. These data are in contra-

INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY


Fig. I. Oil-immersion light microscopy of AF Gram stain

revealing possible spermatozoa among cellular debris.x 1,000. Fig. 3. Scanning electron photomicrograph. The head and

neck regions of spermatozoa identified on AF Gram stainfrom second-trimester genetic amniocentesis specimen.x 5,000.

Fig. 2. Scanning electron microscopic morphology of sper-matozoa identified on AF Gram stain from second-trimestergenetic amniocentesis specimen. 1,000.

distinction to the recently published report of a

5.09% (8/157) incidence of occult AF infection at

the time of second-trimester amniocentesis. 4 Theydo, however, agree with the findings of Thomsenet al., s who reported culturing no M. hominis andhad positive culture for U. urealyticum of 150midtrimester AF specimens.

Genital mycoplasmas are a common isolate inAF obtained from patients in preterm labor both inour laboratory and other laboratories. 1,3 Our priorsuccessful culturing of F. nucleatum and the genitalmycoplasmas in patients with preterm labor sup-ports the accuracy of our culture results. The ab-sence of AF bioactive leukoattractants, previouslyshown to be accurate predictors of preterm deliv-ery, failed tocolysis, and histologic chorioamnioni-

Fig. 4. Scanning electron photomicrograph. The mitochon-drial cristae are seen in the center of the neck region ofspermatozoa identified on AF Gram stain from second-tri-mester genetic amniocentesis specimen, x 50,000.

tis in the preterm labor/intact membrane popula-tion, provides additional supportive evidence thatthe negative cultures were accurate.

The high culture-positive rate and the failure toculture for Mycoplasma sp. and U. urealyticum inthis prior report prompted the current study. Inaddition, the bacteria isolated by Goldstein et al. 4

from second-trimester AF were generally differentfrom those cultured in reported studies on AF frompatients in preterm labor with intact membranes. 1,3

A closer evaluation of the report by Goldstein etal. 4 reveals that 5 of the cultures showed only scant

growth, defined as < 10 colonies/plate. Moreover,

138 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY


whether the aborted pregnancy had scant growth ofEscherichia coli, as reported in the text, or over-

abundant growth, as reported in the table, cannot

be determined. The 3 positive cultures in the pre-vious report with bacterial growth greater than scant

would give an incidence of 3/157 (1%), not statisti-cally different from that reported in the presentstudy.

The documentation of an accurate incidence ofoccult infection in the second trimester is importantwhen considering 1) the possible association withcomplications of amniocentesis and 2) the recom-

mendation by Goldstein et al. 4 that bacteriologicevaluations be carried out on all AF specimensfrom the second trimester, at significant additionalcost and without a recommendation of how to man-

age positive cultures in this setting. The data pre-sented in this study do not support routine bacteri-ologic study of AF from patients presenting forgenetic amniocentesis. The concordance of the bac-teriologic tests and the AF samples evaluated forbioactive leukoattractants supports a low incidenceofoccult AF infection in the midtrimester. Whetherthe difference between the data previously reportedand that presented in the current study is related to

procedure or bacteriologic technique or patient pop-ulation cannot be determined.Of considerable interest is the previously unre-

ported finding of spermatozoa on Gram stain of 2second-trimester AF specimens from patients withintact membranes. Both patients delivered at term

without complications. Sperm is a well-known car-

tier of bacteria;6 and transmembranous movementof sperm, if confirmed, represents a potentiallyimportant etiology for intraamniotic infection.While scanning electron microscopy provides sup-portive evidence for this unexpected finding, thepossibility that this finding represents artifacts ofAF staining techniques cannot be entirely elimi-nated. Further studies are necessary to confirm or

refute this potentially important finding.

REFERENCES1. Romero R, Sirtori M, Oyarzun E, et al.: Infection and

labor. V. Prevalence, microbiology, and clinical signifi-cance of intraamniotic infection in women with pretermlabor and intact membranes. Am J Obstet Gynecol 161:817-824, 1989.

2. Pankuch GA, Cherouny PH, Botti JJ, Appelbaum PC:Amniotic fluid leukotaxis assay as an early indicator ofchorioamnionitis. Am J Obstet Gynecol 161:802-807,1989.

3. Cherouny PH, Pankuch GA, Botti JJ, Appelbaum PC:The presence of amniotic fluid leukoattractants accuratelyidentifies histologic chorioamnionitis and predicts tocolyticefficacy in patients with idiopathic preterm labor. Am JObstet Gynecol 167:683-688, 1992.

4. Goldstein I, Zimmer EZ, Merzbach D, Peretz BA, PaldiE: Intraamniotic infection in the very early phase of thesecond trimester. Am J Obstet Gynecol 163:1261-1263,1990.

5. Thomsen AC, Taylor-Robinson D, Brogaard Hansen K,et al.: The infrequent occurrence of mycoplasmas in amni-otic fluid from women with intact fetal membranes. ActaObstet Gynecol Scand 63:425-429, 1984.

6. Toth A: Alternative causes of pelvic inflammatory disease.

J Reprod Med 28:699-702, 1983.

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